HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ISOLATION, SPECTROSCOPIC CHARACTERIZATION, AND IMMUNOSUPPRESSIVE ACTIVITIES OF 2 RAPAMYCIN DEGRADATION PRODUCTS

A high performance liquid chromatographic method has been developed for the isolation of two degradation products of rapamycin which is currently under development as an immunosuppressive agent. Prior to isolation, the drug was incubated at 37 degrees C in rat bile or ammonium acetate (pH 8.0). The isolation was achieved by a Supelco, PLC-18 21.2 x 250 mm, 18 mu m column using methanol/ammonium acetate gradient mobile phase. After evaporation of methanol, the remaining eluates were lyophilized. The isolated degradation products were characterized by negative ion fast atom bombardment mass spectrometry (FAB MS) and proton nuclear magnetic resonance spectroscopy (H-1 NMR). Degradation product A was found to be a macrolide ring-opened hydrolysis product of rapamycin where the C25 ester bond had been hydrolyzed. Degradation product B was determined to be a ring-opened isomer of rapamycin. B had less than 4% of the potency of rapamycin in a thymocyte proliferation assay, while A had minimal activity at concentrations tested.